Intracellular delivery of therapeutic agents

ABSTRACT

The preparation and use of a transducing polypeptide (TP)— lipid vesicle complex having a small proportion of positively charged (cationic) lipids in the make-up of the lipid vesicle, e.g., liposome, for safe and efficient intracellular delivery of therapeutic agents, such as proteins, DNA, small molecules and/or other drugs, into a cell of a higher organism, in vitro or in vivo is disclosed. The delivery system of the invention results in increased efficacy of intracellular delivery of such agents, bypassing the endocytotic pathway of intracellular delivery while at the same time minimizing the toxicity of the delivery system towards the recipient cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. Provisional Application No.60/356,526, filed Feb. 13, 2002, entitled INTRACELLULAR DELIVERY OFDRUGS AND DNA, the whole of which is hereby incorporated by referenceherein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

N/A

BACKGROUND OF THE INVENTION

Numerous disorders and diseases have been shown to be associated withvarying degrees of genetic impairment, such as point mutations, genedeletions or duplications. Thus, the management of these diseases veryoften requires manipulation at the genetic level, including suchpossibilities as substitution of the malfunctioning gene(s) orintroduction of a multigene complex, usually via the use of “vectors,”which carry a fragment of DNA and are able to replicate within the cellwith consequent expression of the protein in question. This process ofintroducing a gene or DNA into a cell, or transfection, has enormousbiotechnological application, but even more so in the ever wideningfield of medical gene therapy.

Several different methods of DNA delivery into cells are currentlyavailable; however, only a few of these are actually in clinical trialsor use [Roth et al., (1995); Lisziewicz et al., (1995)]. Common methodssuch as co-precipitation, membrane permeabilization and electroporationhave not been applicable for in vivo use [Song et al., (1995)). Viruseshave evolved highly efficient mechanisms of entering cells andreproducing within the host cell. Adenoviruses, retroviruses, and theirfragments are, thus, attractive vehicles for gene therapy since they arehighly stable, exhibit wide tropism, and can infect quiescent as well asdividing cells (Perricaudet et al., (1995)]. Unfortunately, in spite oftheir efficiency, they have limitations, including size limit for DNAincorporation, evocation of an immunological response and potentialtransformation in the host cell [Bett et al., (1993); Yang et al.,(1995); Miller et al., (1990)].

Non-viral vector systems include the bombardment with DNA-coatedparticles, the use of polycations as DNA carriers, and receptor-mediatedgene delivery involving complexing plasmid DNA to specific targetingproteins [Rech et al., (1996); Basu, S. K., (1990); Leamon et al.,(1991)]. Intensive interest has also focused on amphiphiles such ascationic lipids (liposomes/lipoplexes) as vehicles for the transfer ofrecombinant genes into a variety of tissues. Liposomes are well knowndrug carriers with a large capacity for delivering drugs encapsulatedinto vesicles or incorporated into the membrane [Woodle et al.; (1995),Gergoriadis, G., (1995)]. For many years, liposomes have beeninvestigated as a means for gene delivery. However, to associate thegene material, i.e., negatively charged DNA, with liposomes and to makecells capture liposomes by endocytosis, the liposomes have to becomposed using a substantial addition of positively charged lipids, in aquantity which is quite toxic. Commercial Lipofectin® (positivelycharged liposomes) can be rather successfully used for celltransfection, but the concentration of lipids (and, consequently, thequantity of delivered DNA) is critical in this case, again, because ofpotential toxicity problems [Scheule et al., (1997); Dokka et al.,(2000); Xu et al., (1996)]. Moreover, the usual routes ofinternalization of drug and DNA carriers by endocytosis or pinocytosisresult in subsequent and substantial degradation of intracellularlydelivered drugs and DNA by endosomal and lysosomal enzymes, whichstrongly limits the efficacy of transfection as therapy [Frankel et al.,(1988)].

A key limiting factor in the intracellular delivery of drugs and DNA iscell membrane traversability, and numerous approaches have beenattempted to overcome this problem. For example, several membranetranslocating signal peptides have been described. These include the“protein transduction domains” (PTDs) of HIV-1 TAT protein, the VP22herpes virus protein and Antennapedia protein (ANTP) [Fawell et al.,(1994); Vives et al., (1997); Derossi et al., (1994); Phelan et al.,(1998)], which have been shown to efficiently traverse biologicalmembranes. This process is receptor- and transporter-independent, is notendocytosis-mediated and seems to target the lipid layer directly. Manyof these peptides promote lipid membrane-reorganizing processes, such asfusion and pore formation, involving temporary membrane destabilizationand subsequent reorganization [Prochiantz, A., (1999)]. The minimal PTDof the TAT protein comprises residues 47-57. This and similar peptidesderived from TAT protein are termed as TAT peptides. Common structuralfeatures of TAT and ANTP PTDs include the presence of many positivelycharged basic amino acids (arginine and lysine), as well as the abilityto adopt an alpha helical conformation. The use of these peptides andprotein domains with amphipathic sequences for drug and gene deliveryacross cellular membranes is getting increasing attention [Fawell etal., (1994); Lindgren et al., (2000); Wagner, E., (1999); Plank et al.,(1998); Mi et al., (2000)]. Covalent hitching of proteins, smallmolecule drugs or DNA onto PTDs may circumvent conventional limitationsby allowing the transport of these compounds directly into the cytoplasmof a wide variety of cells in vitro and in vivo. For example, TATpeptide chemically attached to various proteins (e.g., horseradishperoxidase and β-galactosidase) was able to deliver these proteins athigh levels to various cells and tissues in the heart, lung and spleenof mice [Schwarze et al., (2000)].

PTDs such as the TAT peptides have been used for intracellular deliveryof drug carriers, such as micelles and nanoparticles. For example,dextran-coated iron oxide colloidal particles about 40 nm in diameterand containing several attached molecules of TAT peptide per particlewere delivered into lymphocytes much more efficiently than freeparticles [Lewine et al., (2000)]. Although DNA modified directly withTAT peptide demonstrated good intracellular localization and a gooddegree of transfection, the direct modification of DNA may beaccompanied by various side-effects [Schwarze et al., (2000); Eguchi etal., (2001); Allinquant et al., (1995)].

The internalization by cells of a truncated HIV-1 TAT protein basicdomain was shown to proceed at 4° C., i.e., not involving the endocyticpathway [Vives et al., (1997)]. An energy-independent mechanism oftranslocation through biological membranes was also found for the 60-merhomeodomain of ANTP and was not abolished by directed mutagenesis withinthe polypeptide C-terminal region; a 16-mer polypeptide derivative withtranslocating activity was also developed. Again, no classicalendocytosis was assumed, since the peptide was effectively internalizedat 4° C. [Vives et al., (1997); Derossi et al., (1996)].Amphipathicity-independent cellular uptake of cell-penetrating peptidesalso has been shown, and this uptake was also temperature-independent[Derossi et al., (1996)]. Although the actual mechanism of thisuptake/translocation has not yet been clearly established, it issuspected that some form of a ligand-lipid interaction plays a role andthat direct contact between the translocating moiety and cell membraneis required [Prochiantz, A., (1999)].

Since traversal through cellular membranes represents a major barrierfor efficient delivery of macromolecules into cells, the TAT peptide mayserve to ferry not only various drugs into mammalian cells in vitro andin vivo, but also larger particles such as liposomes. The efficientintracellular delivery of TAT peptide-modified liposomes was recentlydemonstrated [Torchilin et al., PNAS (2001)]. Yet, even with thisprogress, the positively charged lipids of the known liposome systemspose severe toxicity problems for full therapeutic application. Thus,much more progress is required before the development of an efficientand non-toxic system for delivery of drugs and DNA directly to thecytoplasm and into peri-nuclear or nuclear region, bypassing theendosomal pathway, would be possible.

BRIEF SUMMARY OF THE INVENTION

This invention is directed to the preparation and use of a transducingpolypeptide (TP)-lipid vesicle complex having a small proportion ofpositively charged (cationic) lipids in the make-up of the lipidvesicle, e.g., liposome, for safe and efficient intracellular deliveryof therapeutic agents, such as proteins, nucleic acids, small moleculesand/or other drugs, into a cell of a higher organism, in vitro or invivo. The positively charged lipid is in an amount sufficient forcomplexing with a therapeutic agent but is less than 45 mol % of totallipid in the lipid vesicle, e.g., from 0.01 to 45 mol %, preferably from0.05 to 35 mol %, more preferably from 0.1 to 25 mol %, even morepreferably from 0.5 to 10 mol %, and most preferably about 10 mol %positively charged lipid. Examples of transducing polypeptides include aprotein transduction domain of the HIV-1 TAT protein, the VP22 herpesvirus protein and the Antennapedia protein (ANTP).

To take advantage of the positive charge on the liposome portion of thedelivery system of the invention, it is especially convenient to use asthe therapeutic agent a drug molecule bearing a negative charge at thepH value of the liposome preparation. Smaller pieces of nucleic acids,such as RNA, anti-sense RNA, small interfering RNA (siRNA) ormitochondrial DNA (mtDNA), may also be included in the aqueous bufferduring liposome preparation and will become trapped in the aqueous core.Very large pieces of (negatively charged) DNA usually cannot beincorporated within the liposome vesicle. Such pieces are preferablycomplexed via charge interaction with the positively charged, pre-formedliposomes.

The delivery system of the invention results in increased efficacy ofintracellular delivery of such agents, bypassing the endocytotic pathwayof intracellular delivery while at the same time minimizing the toxicityof the delivery system towards the recipient cells. Also contemplated aswithin the invention are kits for the preparation of delivery systems ortherapeutic compositions according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof and from theclaims, taken in conjunction with the accompanying drawings, in which:

FIGS. 1A-1D are micrographs showing intracellular trafficking ofRh-PE-labeled and FITC-dextran-loaded TATp-liposomes within BT20 cells.Typical patterns of intracellular localization and integrity ofTATp-liposome are shown, after 1 hour (FIG. 1A); 2 hours (FIG. 1B); 4hours (FIG. 1C); and 9 hours (FIG. 1D). On each figure panel, (a)—DIC(differential interference contrast) light; (b)—DIC with a Rh filter;(c)—DIC with a FITC filter; (d)—DIC composite of (a), (b) and (c), allat x400;

FIG. 2A shows gel-electrophoresis results of free pEGFP-N1 plasmid (1),TATp-liposome/pEGFP-N1 complex (2), and Triton X-100-treatedTATp-liposome/pEGFP complex (3); and FIG. 2B shows freeze-etchingelectron microscopy of TATp-liposomes (a) and TATp-liposome/pEGFP-N1complex (b);

FIGS. 3A-3B show cell transfection in vitro with TATp-liposome/pEGFP-N1complexes and TATp-free liposome/pEGFP-N1 complexes. FIG. 3A is a graphdisplaying flow cytometry data (the number of fluorescent cells andfluorescence intensity on the FITC channel, FL-1H, after 72 hours) forNIH/3T3 cells: (1)—fluorescence of cells treated with TATp-freeliposome/pEGFP-N1 complex; (2)—fluorescence of cells treated with anequal quantity (DNA and lipids) of Lipofectin®/pEGFP complex;(3)—fluorescence of cells treated with an equal quantity (DNA andlipids) of TATp-liposome/pEGFP complex. Dotted line shows the positionof the peak auto-fluorescense of non-treated cells (negative control);and FIG. 3B shows micrographs (x400, after 72 hours) of NIH/3T3 (a, b)and H9C2 (c, d) cells treated with TATp-liposome/pEGFP-N1 complex. (a)and (c)—bright field light microscopy; (b) and (d)—epifluorescencemicroscopy with a FITC filter;

FIGS. 4A-4B are bar graphs showing cytotoxicity test results. FIG. 4Ashows the comparative cytotoxicity of low-cationic TATp-liposomesaccording to the invention and Lipofectin® towards NIH/3T3 cells atdifferent lipid concentrations. Incubation was for 24 hrs; cellviability in the presence of 21 μg/ml of TATp-liposomes was taken as100%. FIG. 4B shows the relative viability of NIH/3T3 cells treated withequal quantities (as DNA, at 5 μg) of TATp-liposome/pEGFP-N1 complex andLipofectin®/pEGFP-N1 lipoplex. Incubation was for 4 hrs; cell viabilityin the presence of TATp-liposome/plasmid complex was taken as 100%; and

FIGS. 5 a-5 f are micrographs (x400) of tissue frozen sections from invivo growing LLC tumors in mice showing in vivo transfection withTATp-liposome/pEGFP-N1 complex according to the invention. FIG. 5 a andFIG. 5 b—section from a non-treated tumor (background pattern); FIG. 5 cand FIG. 5 d—section from the tumor injected with TATp-freeliposome/pEGFP-N1 complex; FIG. 5 e and FIG. 5 f—section from the tumorinjected with TATP-liposome/pEGFP-N1 complex. (a), (c), and (e)—brightfield light microscopy after hematoxylin/eosin staining; (b), (d), and(f)—epifluorescence microscopy with FITC filter.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to the preparation and use of a transducingpolypeptide (TP)-liposome complex having a small proportion ofpositively charged (cationic) lipids in the make-up of the liposome forsafe and efficient intracellular delivery of therapeutic agents, such asproteins, DNA, small molecules and/or other drugs, into a cell of ahigher organism, in vitro or in vivo. The delivery system of theinvention results in increased efficacy of intracellular delivery ofsuch agents, bypassing the endocytotic pathway of intracellular deliverywhile at the same time minimizing the toxicity of the delivery systemtowards the recipient cells.

Preparation of liposomes: Lipid vesicles, especially liposomes, may beprepared by any of the commonly used methods known to those of skill inthe art. These include, among others, lipid film hydration, reversephase evaporation and detergent dialysis (see, e.g., LiposomeTechnology, G. Gregoriadis, ed., CRC Press, Boca Raton, vol. 1-3, 1984;Phospholipid Handbook, G. Cevc, ed., Marcel Dekker, Inc., New York,1993; D. D. Lasic, Liposomes. From Physics to Applications, Elsevier,Amsterdam, The Netherlands, 1993).

For example, a lipid film can be formed via solvent evaporation,followed by hydration, using, e.g., cholesterol and phospholipids, suchas the neutral lipid phosphatidylcholine, and from 0.01 to 45 mol %,preferably from 0.05 to 35 mol %, more preferably from 0.1 to 25 mol %,even more preferably from 0.5 to 10 mol %, and most preferably about 10mol % of a positively charged lipid. The lipid combination is dissolvedwith mixing in chloroform, the solvent is removed by vacuum rotaryevaporation, and the resulting lipid film is hydrated in aqueous buffer.This results in the formation of bilayered, membranous, lipid vesiclesof various sizes, shapes and aggregate states. If the liposomes need tobe sized to obtain a homogeneous distribution, they are extruded, priorto the addition of DNA, through 200 nm size polycarbonate filters.Particle size is estimated by a dynamic light scattering technique.

Attachment of transducing polypeptides: Suitable transducingpolypeptides can be directly attached by their reactive groups (such asamino groups, carboxyl groups or sulfhydryl groups) via hydrophobiclinkers (which may be polymers such as polyethylene glycol orpolyvinylpyrollidine) to preformed lipid vesicle (liposome) membranes.Another alternative is to link the peptides to the liposome surface viathe use of a variety of commercially available homo- orhetero-bifunctional reagents known to those of skill in the art (such ascarbodiimide, N-succinimidyl(2′-pyridyldithio)propionate (SPDP) orsuccinimidyl maleidomethyl cyclohexane carboxylate (SMCC), etc.).

For example, as described herein, a TAT peptide (TATp) from the proteintransduction domain of the HIV-1 TAT protein was attached to theliposome bilayer by coupling the amino groups of the peptide top-nitrophenylcarbonyl groups of a linker molecule, such as the polymer(pNP-PEG-PE). This attachment was achieved in two ways. In one method,the linker polymer was included during the formation of the lipid film,the pre-formed liposomes were then incubated with the TATp to allow forcoupling, and unbound TATps were removed by gel filtration. In anothermethod, the TATp was first coupled to the linker polymer, unbound TATpswere removed by dialysis, and the TAT-pNP-PEG-PE was then included inthe lipid film mixture.

Incorporation of therapeutic agents: Therapeutic molecules may beincorporated into the liposomes at different stages of liposomepreparation, depending on the physico-chemical properties of themolecules:

(a) Small molecules that can serve as drugs are usually included in theaqueous buffer during liposome formation. Water soluble compounds aretrapped in the aqueous core of the vesicles, while hydrophobic moleculesdistribute mainly into the lipid bilayer. To take advantage of thepositive charge on the liposome portion of the delivery system of theinvention, it is especially convenient to use a drug molecule bearing anegative charge at the pH value of the liposome preparation, such assulfathiazole, sulfaoxazol, benzylpenicillin, phenobarbital,sulfacetamide, heparin or acidic proteins and peptides (in order tobenefit from the electrostatic attraction to the liposomes). Smallerpieces of nucleic acids, such as RNA, anti-sense RNA, small interferingRNA (siRNA) or mitochondrial DNA (mtDNA), may also be included in theaqueous buffer and will become trapped in the aqueous core.

(b) Very large pieces of (negatively charged) DNA usually cannot beincorporated within the liposome vesicle during lipid film hydration.Such pieces are preferably complexed via charge interaction withpositively charged, pre-formed liposomes. For this purpose, positivelycharged (cationic) lipids, such as DOTMA(2,3-dioleoyloxypropyl-trimetylammonium chloride), a key component ofLipofectin®, and DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), andother positively charged lipids are included in the lipid mixture duringliposome preparation, resulting in an array of positive charges on thesurface of the fully formed liposome.

Most commercial cationic liposome formulations (such as Lipofectamine®or Lipofectin®) require a large amount (50 mol % or more) of cationiclipid in the composition for optimal DNA complexation and transport.However, the transducing polypeptide-liposome delivery system accordingto the invention may be prepared with much less positive charge on thesurface of the liposome that these prior art systems, and therapeuticagents in therapeutic compositions according to the invention may bedelivered into cells of a treated patient with a signficant reduction intoxicity.

The positively charged lipids in the transducing polypeptide-liposomeentity complex with the negatively charged DNA, facilitate DNAassociation, but are not required for the internalization process. Thetransfer of the whole complex through the cell membrane is mediated bythe transducing polypeptides, possibly through the temporary formationof “reversed” micelles in the cell membrane. DNA that can form the abovecomplexes is usually in the form of a plasmid (or, less frequently, anexpression vector) that self-replicates in the transfected cell andexpresses a protein. Alternatively, the DNA to be transported may bechemically attached to the liposome. DNA in certain forms, such assuper-coiled or in small plasmids, may be included inside the liposomes.

In the experiments described herein, the amount of positively chargedlipid was about 10 mol %, with the only goal being to enhance complexformation between the negatively charged DNA and the transducingpolypeptide (e.g., TATp)-liposomes. The quantity of a positively chargedlipid can vary widely, e.g., from 0.01 to 45 mol %, preferably from 0.05to 35 mol %, more preferably from 0.1 to 25 mol %, and most preferablyfrom 0.5 to 10 mol % of total lipid mixture. In a particular case, theexact sub-toxic quantity of such lipid (i.e., the useful upper end ofthe mol % range) can be chosen following the determination of the mol %of positively charged lipid necessary for efficient complex formationbetween the transducing polypeptide-liposome and DNA (i.e., the usefullower end of the mol % range).

The delivery system according to the invention may be loaded with anyappropriate therapeutic agent, and the resulting therapeuticcompositions according to the invention may be administered to a patientorally, topically, or parenterally (e.g., intranasally, subcutaneously,intramuscularly, intravenously, or intra-arterially) by routine methodsin pharmaceutically acceptable inert carrier substances. For example,the compositions of the invention may be administered in a sustainedrelease formulation using a biodegradable, biocompatible polymer, or byon-site delivery using polymeric gels. The therapeutic compositions willbe administered in a dosage appropriate for the therapeutic agent beingadministered. Drug dosages vary widely, e.g., from nanograms perkilogram per day to milligrams per kilogram per day. The delivery systemaccording to the invention is capable of delivering any appropriatedosage desired. Optimal dosage and modes of administration can readilybe determined by conventional protocols.

Also contemplated are kits for the preparation of delivery systems ortherapeutic compositions according to the invention. Such a kit wouldinclude, e.g., transducing polypeptide, pre-modified with linkermolecule for incorporation into the liposome bilayer during liposomeformation; dry lipid film containing an optimal proportion of positivelycharged lipid; and physiological buffer. The components are mixed byshaking or vortexing for several minutes to form the transducingpolypeptide-liposome delivery system according to the invention. Thedelivery system can then be complexed with a plasmid containing theappropriate DNA for the therapeutic treatment contemplated.Alternatively, hydrophobic small molecule therapeutic agents may beincluded in the dry lipid film component of the kit, or hydrophylictherapeutic agents may be included in the buffer component. Then, afterthe components of the kit are mixed, the resulting therapeuticcomposition is ready for administration.

The following examples are presented to illustrate the advantages of thepresent invention and to assist one of ordinary skill in making andusing the same. These examples are not intended in any way otherwise tolimit the scope of the disclosure.

Materials and Methods

Materials

Egg phosphatidylcholine (PC), cholesterol (Ch), phosphatidylethanolamine (PE), polyethylene glycol-PE (PEG-PE), dioleoyltrimethylammonium-propane (DOTAP), and rhodamine-PE (Rh-PE) werepurchased from Avanti Polar Lipids. Para-nitrophenylcarbonyl(pNP)-PEG-PEwas synthesized in-house. FITC-dextran (MW 4400Da), CL-4B Sepharose, andcomponents of buffer solutions were purchased from Sigma. Lipofectin®Reagent was from Invitrogen. TAT-peptide (11-mer:TyrGlyArgLysLysArgArgGlnArgArgArg; MW 1560 Da) was prepared by ResGenInvitrogen Corporation. Cell culture media—RPMI-1640 (RPMI), Eagle's MEM(EMEM), modified Eagle's medium (DMEM), serum-free medium (CompleteSerum-Free), fetal bovine serum (FBS), and heat inactivated FBS weresupplied by Cellgro. Fluorescence-free glycerol-based mounting medium(Fluoromount-G) was from Southern Biotechnologies Associates. A pEGFP-N1plasmid designed for eukaryotic cell expression of the Green FluorescentProtein (GFP) was obtained from Elim Biopharmaceuticals.

Cell Cultures

Human breast adenocarcinoma cells (BT20) were maintained in EMEM (with10 mM pyruvate, non-essential amino acids, L-glutamine, and 10% FBS).Lewis lung carcinoma cells (LLC, established from the lung of a C57BLmouse bearing a tumor resulting from an implantation of primary LLC andwidely used as a model for metastasis and for studying the mechanisms ofcancer chemotherapeutic agents) were maintained in RPMI medium (with 10%FBS). Mouse fibroblasts (NIH/3T3, a continuous cell line of highlycontact-inhibited cells, which was established from NIH Swiss mouseembryo cultures in the same manner as the original random bred 3T3 andthe inbred BALB/c 3T3; the established NIH/3T3 line was subjected tomore than 5 serial cycles of subcloning in order to develop a subclonewith morphologic characteristics best suited for transformation assays)and rat cardiomyocytes (H9C2 myoblasts, a subclone of the originalclonal cell line derived from embryonic BD1X rat heart tissue exhibitingmany of properties of skeletal muscle) were maintained in DMEM (with 10%FBS). Cell lines were from the American Type Culture Collection.

Synthesis of pNP-PEG-PE

pNP-PEG-PE was synthesized according to a published procedure [Torchilinet al., BBA (2001)]. Briefly, 0.1 mmol of PE was dispersed in 8 ml ofchloroform supplemented with 2 ml of triethylamine. The resultingmixture was supplemented with 0.5 mmol of PEG₃₅₀₀-(pNP)₂ in 20 ml ofchloroform and incubated overnight at room temperature under argon.Organic solvents were removed under vacuum. Dried lipid was dispersed in0.01 M HCl and purified by gel filtration on CL-4B Sepharose using 0.01M HCl as an eluent. Pooled fractions containing pNP-PEG-PE werefreeze-dried, dissolved in chloroform and stored at −80° C.

Preparation of TATp-Liposomes

A lipid film was prepared by rotary evaporation from a mixture of PC,Ch, and pNP-PEG-PE (7:3:0.05 molar ratio) with traces of Rh-PE inchloroform. This film was re-hydrated in a citrate buffer pH 5.0,vortexed for 5 min, and then extruded through a polycarbonate filter(pore size 200 nm) using an Avanti Mini-Extruder. When loading withFITC-dextran was required, the latter was added as a component of there-hydration solution. The attachment of TATp to pNP-groups on theliposome surface was carried out by incubating TATp with liposomes in aborate buffer, pH 8.5, overnight at room temperature. The slightlyalkaline conditions allow for the coupling reaction with slow hydrolysisof unreacted pNP residues [Torchilin et al., BBA (2001)]. FreeFITC-dextran and TATp were removed using a Bio-Gel A15M column.Alternatively, pre-synthesized TAT-PEG-PE conjugate was added to thestarting lipid mixture for liposome preparation. The size ofFITC-dextran-loaded Rh-labeled TATp-liposomes was measured by dynamiclight scattering (DLS) on a Coulter N4 Plus Submicron Particle Analyzer(Coulter Electronics).

Gel-Electrophoresis

Electrophoresis was performed using the E-Gel electrophoresis systemfrom Invitrogen Life Technologies. A pre-cast 0.8% E-Gel cartridge waspre-run for 2 min at 60 V, 500 mA followed by loading of 1 μg of DNAsamples in loading dye. Gel running time was approximately 50 min at 60V, 500 mA. The gel was then photographed over an UV box (PhotodyneTechnologies).

Freeze-Fracture Electron Microscopy

The sample was quenched using the sandwich technique and liquidnitrogen-cooled propane. A cooling rate of 10,000 Kelvin per secondavoids ice crystal formation and artifacts possibly caused by thecryofixation process. The fracturing process was carried out in JEOLJED-9000 freeze-etching equipment (Jeol Inc.) and the exposed fractureplanes were shadowed with Pt for 30 sec at an angle of 25-35 degrees andwith carbon for 35 sec (2 kV, 60-70 mA, 1×10⁻⁵ Torr). The replicas werecleaned with fuming HNO₃ for 24-to-36 hours followed by repeatedagitation with fresh chloroform/methanol (1:1 by vol) at least 5 times,and examined at a JEOL 100 CX electron microscope.

EXAMPLE I Intracellular Trafficking of TATp-Liposomes

Intracellular trafficking and localization of TATp-liposomes were testedin BT20 cells grown on coverslips in 6-well plates. At approximately60-70% confluency, cells were incubated with liposomes in a serum-freemedium at 37° C. under 6% CO₂. The medium was removed and the cellswashed with sterile PBS, pH 7.4, after 1, 2, 4, 9 and 24 hr incubation.Coverslips were mounted cell-side down with fluorescence-freeglycerol-based mounting medium and viewed by epi-fluorescence microscopy(Nicon Eclipse E400, Nicon Co.) and deconvolution differentialinterference contrast (DIC) microscopy with pseudo-coloring (Axioplan 2,Zeiss Co.).

Free FITC-dextran showed only minimal intracellular accumulation in theBT20 cells used (not shown), while 200 nm Rh-labeled TATp-liposomesloaded with FITC-dextran rapidly translocated into these cells. Typicalpatterns of time-dependent distribution of TATp-liposomes insideindividual cells are shown in FIGS. 1A-1D. After 1 hour, their diffuselocalization within the cell cytoplasm was evident (FIG. 1A).Intracellular liposomes apparently remained intact within this timeperiod, since the flourescence of the intraliposomal (FITC-dextran) andmembrane (Rh-PE) labels coincided. With time, TATp-liposomes, similar toTATp (37,44), gradually migrated closer to the nuclei, and after 2 and 4hours, a significant fraction of TATp-liposomes was seen surrounding theperi-nuclear region, with a reduced cytoplasmic distribution (FIGS. 1Band 1C). At 9 hours, the degradation of liposomes was observed (diffuseorange/red fluorescence in the cytoplasm and nucleus) with someliposomes remaining in the peri-nuclear region. However, by this timethe FITC-dextran was almost totally released (diffuse greenfluorescence) (FIG. 1D). By hour 24, virtually no internal or membranelabel could be seen inside the cells.

These experiments clearly showed that, in good agreement with earlierobservations [Torchilin et al., PNAS (2001)], the uptake ofTATp-liposomes is fast and efficient. Because of the nuclear tropismimparted by the TATp [Vives et al., (1997); Truant et al., (1999)] TATpconjugation allows for a gradual peri-nuclear localization of liposomes,bypassing the endocytic pathway. Eventually, liposomes are destroyed andrelease their contents. The relatively slow peri-nuclear accumulation ofTATp-liposomes compared to free TATp may be explained by hindereddiffusion of larger liposomal particles in the cytoplasm.

EXAMPLE II Preparation of TATp-Liposome/Plasmid Complexes

Liposomes for complexation with DNA did not contain any fluorescentlabels, but did contain up to 10 mol % of the cationic lipid DOTAP toenhance plasmid association. Liposomes from a mixture of PC, Ch, DOTAP,and pNP-PEG-PE (7:3:1:0.05 molar ratio) were prepared as above, andincubated with the pEGFP-N1 plasmid overnight at 4° C. In a typicalcase, the liposome/plasmid complex containing a total of 2 mg lipid and200 μg DNA was incubated with an appropriate amount of TATp overnight atpH 8.5 in a borate buffer, and purified by gel filtration on Bio-GelA-1.5. The post-column fraction was subjected to agarose gelelectrophoresis to test for the presence and intactness of the plasmidin complex with the liposomes. To determine DNA content, the post-columnTATp-liposome/plasmid complex-containing fraction was treated withTriton X-100 for 1 hour at 37° C. to release the plasmid from thecomplex, and then subjected to agarose gel electrophoresis.Lipofectin®/pEGFP-N1 complex was prepared according to themanufacturer's instruction (Invitrogen Corp.) using same quantities andratios of lipid and DNA (which are within the recommended limits forthis preparation).

TATp-liposomes with a relatively low (≦10 mol %) content of a cationicDOTAP effectively complexed and firmly retained intact plasmid asevidenced by the gel electrophoresis data (FIG. 2A).TATp-liposome/pEGFP-N1 complexes could not enter a gel because of theirlarge size (FIG. 2A, line 2). However, after Triton-X100 treatment, allcomplexed DNA was released in a free form, resulting in a band withintensity close to the control free DNA (compare lines 1 and 3 on FIG.2A). Depending on the particular need, the total quantity of DNA in asample could vary, e.g., from 0.05 to 0.2 μg per 1 μg of lipid, which issimilar to what is normally achieved with Lipofectin® (according tomanufacturer's instructions). Complexation of a plasmid with liposomesonly moderately increased their size (from ca. 150 to ca. 200 nm byDLS). The freeze-etching electron microscopy also showed that the majorfraction of TATp-liposome/plasmid complexes maintained an essentiallyspherical shape with a size of about 200 nm (FIG. 2B). Both preparationsdisplayed convex and concave fracture planes typical of liposomalstructure. The complexes were non-aggregated. Their boundaries appearedsometimes dotted—a phenomenon also observed in other liposome/nucleicacid complexes [J{haeck over (a)}{haeck over (a)}skel{haeck over(a)}inen et al., (1998)]. Thus, TATp-liposomes with a low content of acationic lipid can complex and retain substantial quantities of DNA.

EXAMPLE III Transfection In Vitro

NIH/3T3 or H9C2 cells grown to 60-70% confluency on coverslips wereincubated in serum-free media with TATp-liposome/plasmid complexes orliposome/plasmid complexes (in the quantity required to deliver 5 μg DNAper 200,000 cells at DNA concentration of 0.3 μg/μl of added liposomalsuspension) for 4 hrs at 37° C. under 6% CO₂. The same quantity ofLipofectin®/pEGFP-N1 complex with the same lipid-to-DNA ratio was usedas the control. After incubation, the medium was removed and the cellswere washed twice with sterile PBS and re-incubated with complete DMEMcontaining 10% FBS for 72 hrs. For flow cytometry (FACScan™, BectonDickinson Biosciences), NIH/3T3 cells were grown in 25 cm² flasks andfixed in 4% paraformaldehyde. GFP expression was visualized by lightmicroscopy and epifluorescence microscopy using a FITC filter.

The results of the treatment of NIH/3T3 fibroblasts and H9C2cardiomyocytes with TATp-liposome/pEGFP-N1 complexes are presented inFIGS. 3A-3B. The flow cytometry data (FIG. 3A) show that the treatmentof NIH/3T3 cells with TATp-free liposome/pEGFP-N1 complexes results in aslight increase in cell fluorescence (compare the position of the curve1 peak on FIG. 3A with the dotted line showing the peak autofluorescenceon non-treated cells). This fluorescense may result from some celltransfection via non-specifically captured plasmid-bearing liposomes. Atthe same time, cells treated with TATp-liposome/pEGFP-N1 complexesdemonstrated a substantially higher fluorescence (curve 3 vs. curve 1 onFIG. 3A), i.e., a higher transfection outcome. At similar conditions,Lipofectin®/pEGFP-N1 complexes provided essentially the same extent oftransfection and fluorescence level as TATp-liposome/plasmid complexes(curve 2 on FIG. 3A).

Confocal microscopy confirmed the transfection of both NIH/3T3 and H9C2cells with TATp-liposome/DNA complexes (FIG. 3B). From 30 to 50% of bothcell types in the field of view show a bright green fluorescence, whilelower fluorescence was observed in virtually all cells. As can be seenfrom the photographs, the transfection was not accompanied by anyvisible toxic effects. All cells appear morphologically normal.

EXAMPLE IV Cytotoxicity Assay

NIH/3T3 cells were seeded in 96-well tissue culture microtiter plates.After 24 hrs, the culture medium was removed and the cells were treatedwith TATp-liposomes, Lipofectin®, TATp-liposome/pEGFP-N1 complex, orLipofectin®/pEGFP-N1 complex in serum-free medium. The experiments werecarried out both in the absence of the plasmid at several differentconcentrations of low-cationic TATp-liposomes and Lipofectin®, and inthe presence of plasmid, at concentrations of TATp-liposomes andLipofectin® required to provide DNA concentration of 5 μg/ml (totallipid concentration for both preparations varied from approximately 20to approximately 100 μg/ml). After 24 hrs in case of plasmid-freepreparations and after 4 hrs in case of DNA-containing preparations, themedium was removed, CellTiter 96 Aqueous One solution (Promega) added toeach well, and the plates re-incubated for 4 hrs. This assay is based onthe bioreduction of MTS tetrazolium compound (Owen's reagent) into acolored soluble formazan product. The viability of cells was measuredusing a plate-reader (Multiscan MCC/340, Fisher Scientific) at 490 nm.Relative viability was calculated with cells treated only with mediumalone as a control. The statistical treatment of the data was performedaccording to the Student's T test for two populations.

As can be seen in FIG. 4A, at similar concentrations, low-cationicTATp-liposomes with 10 mol % of DOTAP were non-toxic for the cells evenafter 24 hrs of incubation, while the same quantities of Lipofectincaused the death of 35 to 65% of cells in a concentration-dependentfashion. This observation was true for any lipid concentration over therange between 20 and 80 μg/ml. In the experiment performed withTATp-liposome/plasmid complex using the same quantity ofLipofectin®-plasmid lipoplex as a control (5 μg DNA and 20 μg of lipidper ml in both cases, which is a normal working concentration for theLipofection procedure), the TATp-liposome/plasmid complex was about 25%less toxic than the Lipofectin®-plasmid lipoplex to the NIH/3T3 cellsafter only 4 hrs of incubation (FIG. 4B). Thus, complexes according tothe invention of DNA with TATp-liposomes with a low content of apositively charged lipid can complex and deliver DNA into cells withless toxic effects than is typical for many non-viral DNA deliverysystems with a high content of positive charge.

EXAMPLE V Transfection In Vivo

The aim of this study was to carry out an in vivo transfection in awhole animal model with Lewis lung carcinoma cells (LLC). Transfectionwas localized by direct administration of TATp-liposome/pEGFP-N1complexes into the tumor tissue to minimize the non-specifictransfection of other tissues.

LLC tumors were grown in C57BL/6 mice (Charles River Laboratories) bysubcutaneous injection of 8×10⁴ LLC cells per mouse into the left flank(protocol 011022 approved by the IACUC, Northeastern University, Nov.26, 2001). Tumors were injected at 4-5 different spots with 100 μl ofTATp-liposome/pEGFP-N1 complex in HBS, pH 7.4, after they reached5-to-10 mm in diameter. Mice were sacrificed at 72 hr later by cervicaldislocation, and excised tumors were fixed in a 4% bufferedparaformaldehyde overnight at 4° C., blotted dry of excessparaformaldehyde and kept in 20% sucrose in PBS overnight at 4° C.Cryofixation was done by immersion of tissues in ice cold isopentane for3 min followed by freezing at −80° C. Fixed, frozen tumors were mountedin Tissue-Tek OCT 4583 compound (Sakura Finetek USA) and sectioned on aLeica Jung Frigocut 2800N (Leica Instrument). Sections were mounted onslides and analyzed by fluorescence microscopy and withhematoxylin/eosin staining. Tumor-bearing mice injected with TATp-freeliposome/plasmid complexes of the same composition and non-injected micewith similar-sized tumors were used as negative controls.

FIG. 5 presents the in vivo results with LLC-bearing mice.Histologically, hematoxylin/eosin-stained tumor slices in both controland experimental animals showed a typical pattern of poorlydifferentiated carcinoma (polymorphic cells with basophilic nucleiforming nests and sheets and containing multiple sites ofneoangiogenesis; FIGS. 5 a, 5 c, and 5 e). Under the fluorescencemicroscope, samples from control mice (non-treated mice or mice injectedwith TATp-free liposome/plasmid complexes; FIGS. 5 b and 5 d) showedonly a background fluorescence, while slices from tumors injected withTATp-liposome/plasmid complexes contained bright green fluorescence intumor cells (FIG. 5 f) indicating the TATp-mediated transfection invivo.

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While the present invention has been described in conjunction with apreferred embodiment, one of ordinary skill, after reading the foregoingspecification, will be able to effect various changes, substitutions ofequivalents, and other alterations to the compositions and methods setforth herein. It is therefore intended that the protection granted byLetters Patent hereon be limited only by the definitions contained inthe appended claims and equivalents thereof.

1. A delivery system for a therapeutic agent, said system comprising: alipid vesicle complexed with one or more transducing polypeptides, saidlipid vesicle comprising positively charged lipid, wherein saidpositively charged lipid is in an amount sufficient for complexing witha therapeutic agent but less than 45 mol % of total lipid in said lipidvesicle.
 2. The delivery system of claim 1, wherein said lipid vesiclecomprises between 0.01 and 45 mol % positively charged lipid.
 3. Thedelivery system of claim 1, wherein said lipid vesicle comprises between0.05 and 35 mol % positively charged lipid.
 4. The delivery system ofclaim 1, wherein said lipid vesicle comprises between 0.1 and 25 mol %positively charged lipid.
 5. The delivery system of claim 1, whereinsaid lipid vesicle comprises between 0.5 and 10 mol % positively chargedlipid.
 6. The delivery system of claim 1, wherein said lipid vesiclecomprises about 10 mol % positively charged lipid.
 7. The deliverysystem of claim 1, wherein said transducing polypeptide is a proteintransduction domain of the HIV-1 TAT protein.
 8. The delivery system ofclaim 1, wherein said transducing polypeptide is the VP22 herpes virusprotein.
 9. The delivery system of claim 1, wherein said transducingpolypeptide is the Antennapedia protein (ANTP).
 10. The delivery systemof claim 1, wherein said positively charged lipid is2,3-dioleoyloxypropyl-trimetylammonium chloride or1,2-dioleoyl-3-trimethylammonium-propane.
 11. The delivery system ofclaim 1, wherein said transducing polypeptide is complexed with saidlipid vesicle via a linker molecule.
 12. The delivery system of claim11, wherein said linker molecule is a polymer.
 13. The delivery systemof claim 11, wherein said linker polymer is attached to said lipidvesicle during lipid vesicle formation.
 14. A therapeutic compositioncomprising the delivery system of claim 1; and a therapeutic agentcomplexed with said delivery system.
 15. The therapeutic composition ofclaim 14, wherein said therapeutic agent is nucleic acid.
 16. Thetherapeutic composition of claim 15, wherein said nucleic acid is DNA.17. The therapeutic composition of claim 16, wherein said DNA is in theform of a plasmid.
 18. The therapeutic composition of claim 16, whereinsaid DNA is in the form of an expression vector.
 19. The therapeuticcomposition of claim 14, wherein said therapeutic agent is RNA.
 20. Thetherapeutic composition of claim 14, wherein said therapeutic agent isanti-sense RNA.
 21. The therapeutic composition of claim 14, whereinsaid therapeutic agent is small interfering RNA.
 22. The therapeuticcomposition of claim 14, wherein said therapeutic agent is mitochondrialDNA.
 23. The therapeutic composition of claim 14, wherein saidtherapeutic agent is a small molecule drug.
 24. The therapeuticcomposition of claim 14, wherein said therapeutic agent is complexedwith the external surface of said lipid vesicle.
 25. The therapeuticcomposition of claim 14, said lipid vesicle having an aqueous core, andwherein said therapeutic agent is in said aqueous core.
 26. Thetherapeutic composition of claim 14, said lipid vesicle having a lipidbilayer membrane, and wherein said therapeutic agent is distributed insaid lipid bilayer membrane.
 27. A method of treating a patient in needof therapy, said method comprising providing a patient in need oftherapy; and administering to said patient a therapeutically effectiveamount of the therapeutic composition of claim
 14. 28. A kit forpreparing a delivery system for a therapeutic agent, said kit comprisinga transducing polypeptide; a mixture of dry lipids, said mixturecomprising positively charged lipid, wherein said positively chargedlipid is in an amount sufficient for complexing with a therapeutic agentbut less than 45 mol % of total lipid in said lipid vesicle; andphysiological buffer.
 29. The kit of claim 28, wherein said transducingpolypeptide is modified with a linker molecule.
 30. A kit for preparinga therapeutic composition, said kit comprising the kit of claim 28,wherein said dry lipid mixture comprises a hydrophobic therapeuticagent.
 31. A kit for preparing a therapeutic composition, said kitcomprising the kit of claim 28, wherein said physiological buffercomprises a hyrophylic therapeutic agent.